Host-microbe interactions, both beneficial and pathogenic, are a rapidly developing area of research with important implications for understanding the normal microbiota, as well as its disturbance by infection. The monospecific symbiosis between the bioluminescent bacteria Vibrio fischeri and the Hawaiian bobtail squid, Euprymna scolopes, provides us with an ideal model with which to study the earliest events of symbiotic initiation. This proposal will focus on a key bacterial signaling pathway, chemotaxis, which allows a bacterium to recognize and respond to changes in its environment. Characterization of bacterial chemotaxis in the context of a beneficial host-microbe association will provide a more complete picture of how microbes interact with and respond to the complex environments presented by host tissues, while migrating from the ambient seawater into a host organism. The overall goals of this research are to characterize the methyl-accepting chemotaxis proteins (MCPs) which act as the ligand receptors in the bacterial chemotaxis machinery, as well as the regulation of these receptors. MCPs have been well studied to date in E. coli. However, E. coli only uses a set of five of these receptors, while the majority of bacteri are known to have many more. V. fischeri is predicted to encode 43 MCPs, and their expression is known to be relevant to colonization of the host. The colonization of E. scolopes by V. fischeri has been shown to occur in several distinct stages, and involves the bacterium migrating through several environments and tissues as it moves from the bacterioplankton to the interior light organ of the animal. This research will further clarify the specific ligands recognized by V. fischeri, and which of these are involved during the initiation of symbiosis. The first aim will address the receptor-ligand interactions, and will be pursued using established techniques such as soft-agar motility assays and capillary-tube migration assays, as well as a FRET-based assay that allows investigation of specific ligand-protein interactions in a receptorless- background strain of E. coli. The second aim will investigate the regulation of these MCPs, and will utilize both transcriptional profiling by RNAseq and fluorescent reporter methods, in experiments designed to determine whether distinct stages of the colonization involve specific receptors. The tools used and developed during the course of this project will also be applicable to other studies, both in the Ruby lab and in other labs studying host-microbe interactions. This project will also provide significant training to the post-doctoral fellow in several areas, includng advanced microscopy and transcriptional profiling techniques.